Project Summary Alternative splicing is an important cellular process that is highly regulated and impacts many fundamental cellular decisions and we only have a rudimentary understanding of how it is regulated, given the range of competing factors. The binding of RNA binding proteins (RBPs) to specific sites in pre-mRNAs plays a central role in regulating alternative splicing. Changes in the concentrations of RBPs that regulate splicing have been implicated in many human diseases including cancer, heart and neurological disease. To understand how alternative splicing is regulated over a concentration gradient, novel cellular dosing systems have been created with the splicing factor MBNL1. The MBNL1 dosing systems can be used to study alternative splicing up to a 20-fold range of protein. Next generation sequencing and global RNA structure probing approaches are being used to characterize how changes in MBNL1 concentration control alternative splicing. The biological implications of these studies are many and significant. The results from the MBNL1 dosing studies will provide a framework to understand and potentially predict splicing events that will change at low MBNL concentrations and do so quickly (steep slope) in different tissues during development. The dosing system can also be used to predict and model the behavior of splicing events in which the concentration of MBNL1 is altered and will help guide the selection of biomarkers for myotonic dystrophy.